We have used affinity-purified antibodies to turkey gizzard smooth muscle myosin light chain kinase to study the immunological properties of these enzymes. Immunocytochemical experiments on muscle tissues demonstrated that these antibodies only reacted with antigenic determinants found in smooth muscles, suggesting that smooth and striated muscle myosin light chain kinases are immunologically distinct. Immunoprecipitation experiments demonstrated that avian and canine smooth muscle myosin light chain kinases have molecular weights of 130,000 and 150,000, respectively. The following results were obtained from experiments on myosin light chain kinases purified from turkey gizzard and bovine tracheal smooth muscles and human platelets: (1) a precipitin band only when the antibodies are cross-reacted with the turkey gizzard enzyme; (2) the production of peptides with different molecular weights when the three enzymes are digested with the S. aureus V8 protease and differential binding of the antibodies to these peptides; (3) the binding of only about a third of the antibodies to the tracheal enzyme when compared to the binding of antibodies to the gizzard enzyme on an Elisa assay; (4) the inhibition of the catalytic activity of all three kinases at approximately the same antibody:kinase ration. These data demonstrate that myosin light chain kinases are an immunologically heterogeneous group of proteins. They also suggest that the smooth muscle and platelet enzymes may have originated from a common genetic ancestor and that regions required for catalytic activity are conserved in all three enzymes despite substantial changes that appear to have occurred in other regions of the enzymes.